1. Field of the Invention
The present invention relates to a method for structural analysis of an organic compound using a high-energy collisional-induced dissociation-mass/mass spectrum (hereinafter, simply referred to as CID-MS/MS spectrum). Especially, the present invention relates to a method for causing a charge-remote fragmentation with respect to a nitrogen-containing organic compound and a reagent to be used in such a method.
2. Description of the Background Art
Various kinds of physical and chemical methods have been applied as means for structural analysis and identification of organic materials with unknown or presumptive chemical formulas. In a most commonly accepted way in the prior art, for example, a target organic compound is subjected to the analysis using an infrared absorption spectrum (IR spectrum), a proton- or carbon-nuclear magnetic resonance spectrum (H1- or C13-NMR spectrum), a mass spectrum (MS spectrum), an X-ray crystal diffraction or the like, followed by the analysis of the resulting data to determine or identify a chemical formula of the target organic compound, and so on.
These means for structural analysis of organic compounds have been remarkably developed together with scientific and technological advances. For example, a MS/MS method is becoming one of the methods frequently used for advanced structural determination of peptides as nitrogen-containing organic compounds. In general, such a MS/MS method is a combination of an ionization method such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization with a method of collisional-induced dissociation (CID) that permits molecular dissociation with low energy or post-source decay (PSD).
A MS/MS spectrum to be used in the MS/MS method has several advantages because it is highly sensitive and is measurable in a mixture. Therefore, the MS/MS method is widely used as a method for determining a primary sequence of peptide. In many cases, however, observed sequence ions (dissociated ions) complicate the structural analysis of a target compound. In the case of peptides, for example, the structural analysis can be complicated by the presence of undesired ion peaks generated in a variety of cleavage fashion that usually accompanies with bond-cleavage between carbon-hetero atom bonds. As other advantages, the MS/MS method is capable of obtaining information about the differences in molecular weights corresponding to amino acid residues that make up the target peptide. However, for example, the MS/MS method is difficult to distinguish leucine (Leu) and isoleucine (Ile), which are isomers with respect to each other. In addition, the MS/MS method is also difficult to distinguish glutamine (Gln) and lysine (Lys) which are of the same molecular weight.
For arginine (Arg)- or lysine (Lys)-rich peptides, other basic groups, specifically guanidine groups or amino groups may be also included in their amino acid structures. Thus, the MS/MS method further complicates the structural analysis because of causing a complicated spectrum as a result of dispersing charge sites.
Recently, for solving the above disadvantages, attention is being given to the MS/MS analysis that utilizes charge-remote fragmentation (CRF) to be obtained from high-energy collisional-induced dissociation (CID).
More specifically, the CRF is characterized in that CRF can be observed by performing a molecular ionization using a first-stage mass spectrometer (MS) by introducing a sulfonic acid residue on the molecular end of peptides, especially on N-terminal thereof, or by holding (charging) a polar functional group such as a quaternary ammonium salt residue on the N-terminal, followed by performing forcible fragmentation, i.e., high-energy collisional-induced dissociation (CID).
The CRF observed here is characterized in that unidirectional bond cleavage between not only carbon-hetero atom bonds but also carbon-carbon bonds. Moreover, the CRF occurs from a distant place of a sulfonic acid group or a polar functional group such as a quaternary ammonium salt, which exists on the molecular end, and fragmentation arises regularly in the direction perpendicular to a molecular axis.
Here, the basic fashion of dissociation in which the CRF can be observed will be explained with reference to the following schematic formula. In this schematic formula, each of lower-case letters of the alphabet, a, b, c, d, e, and f, represents each dissociation site of the molecule. These dissociations occur in alphabetical order, i.e., in order of decreasing the distance from the polar function group. Therefore, peaks of product ions that correspond to these dissociations can be specifically observed.

The MS/MS method is capable of obtaining structural information of the target compound with high accuracy, compared with the above fragmentation at low energy in which fragmentations from both ends of the target molecule can be observed. Therefore, the CID-MS/MS method exerts its greatest force on the structural microanalysis of organic long-chain compounds such as polyamines and polyether organic molecules, notably brevetoxin.
For applying such a structural analysis on the analysis of peptide structure, there is a need for performing the selective introduction of a polar functional group such as a sulfonic acid residue into the N-terminal of peptide. Alternatively, there is a need of the conversion of such an N-terminal into a quaternary ammonium salt residue or the like. However, some of peptides, e.g., mastoparan, bradykinin, insurin, etc., involve the basic amino acid residues on their side chains that may be arginine (Arg), lysine (Lys), ornithine, and diamino-propionic acid. Therefore, it is difficult for the person skilled in the art to perform selective sulfonation only on the N-terminal of each of these compounds or to convert such an N-terminal into a quaternary ammonium form.